(Last updated: April 2020)

The Agilent Bravo, BenchCel, and PlateLoc machines work together as a system: the Bravo functions as a liquid handler, the BenchCel moves plates onto the Bravo and around in the stackers for high-throughput workflows, and the PlateLoc seals plates at the end of the protocol.

The DRSC primarily uses the Bravo to stamp out assay-ready 384-well plates from four 96-well quadrant plates. However, the Bravo has other capabilities as a liquid handler as well and it is possible this robot will be adapted to other protocols in the future.

Stamping out assay-ready plates

1. Take out all the quadrant plates and let them thaw. Vortex for 30 seconds and spin down for 1 minute.

2. The Bravo is controlled by the adjacent Windows computer. Double click the “Bravo” account; there is no password.

3. The Bravo program is called VWorks. Click that icon on the desktop.

• Username: a
• There is no password, just hit enter.

4. As the program loads, make sure the Bravo and the BenchCel are both turned on. The Bravo has a switch on the upper right side of the machine, while the BenchCel has a switch behind the machine to the right, where the power cords are located. Both machines should light up near the top when they are on.

• On the BenchCel, the light-up buttons near the top of the machine are the stacker release buttons. Press a button to release the corresponding stacker from the machine and remove it completely. Wait for the light around the button to stabilize before placing the stacker back into position.

5. Depending on which assay-ready plate will be used and how many copies are being made, there is a plethora of protocols to choose from. The protocols are all located in the following folder: Computer > Local Disk (C:) > Program Files > Agilent Technologies > Protocols > Rong (Tip250). These protocols are organized by plate type and number of plates to be stamped. Select the one that matches your needs.

6. If you do not see the desired protocol, there is a good chance that it is the “Protocols” folder. Hit “back” once to exit the “Rong (Tip250)” folder and scroll down. There is an enormous list of protocols in the “Protocols” folder. Select the one that matches your needs. These protocols do not have the correct tips assigned, so you will have to change the tip type in the protocol.

• Double-click the file to open the protocol in VWorks. The steps of the protocol should be listed. In the expandable sections called “tip box [#],” make sure the tip box listed in parentheses is “96 V11 LT250 Tip Box 1947.002.”
  • If this is not the type of tips listed, expand the tip box section and click the “tip box #” icon.
  • To the right of the screen, the “Task parameters” window will appear. Change the “Plate type” to “96 V11 LT250 Tip Box 1947.002.”
  • Go to “File” > “Save As” and save the protocol in the “Rong (Tip250)” folder.

7. Before running the protocol, always click through to make sure the correct labware is being used. Expand the “Bravo SubProcess” sections of the “Process” step to check that the volume in the “Aspirate” and “Dispense” steps is 5uL. Also check the “Distance from well bottom (0-100)” and make sure that number is either 0.5 or 1. All this information can be found in the “Task parameters” of the corresponding step.

8. To check the parameters of labware, click on “Tools” near the upper left corner, then select “Labware Editor…” This will bring up a window with all labware listed, along with their dimensions and descriptions. We generally use the following labware:

• U-bottom plates: !96 CoStar PP Clr Rnd Well U Btm
• White solid: !384 Corning PS White flt btm 3570
• Black clear: !384 Corning Blk PS Clr Btm
• Evotec: 384 EvoTec PS Blk flt btm
• Tips: 96 V11 LT250 Tip Box 1947.002

9. After making any necessary edits to the protocol, click the “Simulation is off” button with the blue gear towards the top of the window. The button will now say “Simulation is on,” allowing a dry run of the protocol to make sure the protocol runs in theory.

• Hit “Start” with the play button. Click through the next few windows by hitting “Next” until coming across “Finish” on the comments window. These messages are mostly about scheduling and annotating the run, which is usually unnecessary.
• The dry run will proceed. If any errors appear, make the appropriate adjustments. If there are no errors, the program will show that the protocol dry run has concluded.

10. After the dry run, set up the Bravo deck with tip boxes and quadrant plates.
 

• Positions 1, 2, 3, and 9: tip box. The tip boxes we use are the Fluotics AGI-250|F (Agilent LT Head compatible). These can transfer up to 250uL in volume.
• Position 4: quadrant plate 1
• Position 5: quadrant plate 2
• Position 7: quadrant plate 3
• Position 8: quadrant plate 4
• Leave position 6 empty; this is where the BenchCel will deposit plates.

11. Load the assay-ready plates into the left stacker of the BenchCel by slowly lowering them down the stacker from the top. Make sure they are oriented correctly, with the letters on the left of the plate and the numbers going across the top.

12. Check to see if the correct platform is loaded on the PlateLoc. We generally use 290 for Bravo runs.

13. Turn off the simulation in VWorks by clicking the same simulation button (“Simulation is on”). Double check the Bravo deck to make sure all tip boxes and plates are in place at the correct locations. Then hit “Start” to run the program. Click through all windows as with the dry run and hit “Finish.”

14. While the Bravo is running, watch the machine to make sure that everything is going smoothly. Take special care to see if a uniform volume is aspirated from the quadrant plates and completely dispensed into the assay-ready plates.

• Be careful not to get in the way of the Bravo or the BenchCel. There is no safety feature on the robot and getting whacked by metal can hurt.
• Hit the red emergency button if anything goes wrong to immediately stop the machine. If the issue is not urgent, hit “Pause” on the top of the program screen instead. The Bravo will pause after completing the current step.
• Do not use the PlateLoc to seal other plates during a run.
• Do no interfere with the Bravo (barring emergencies) until the program seals all assay-ready plates and stacks them in the right-hand stacker.

15. After the Bravo completes the protocol, a window will pop up on VWorks: “Release stacker racks used in protocols?” Hit “Yes.”

16. Remove the quadrant plates from the deck and seal using the PlateLoc. Store the quadrant plates and the assay-ready plates at -80. Throw tips and their boxes into the biohazard bin.

Notes

• If the Bravo is having motor issues (ex. if the multichannel head is not moving smoothly, or if an error pops up on screen), use the Diagnostics tool to home the multichannel head along the x-, y-, and z-axes. The Diagnostics tool can be found next to the Simulation button on the menu at the top of the window.
• Sometimes when running any protocol that stamps out two assay-ready plates, the Bravo becomes confused and will pause in the middle of the protocol to avoid a deadlock, or running out of space to put plates. If the Bravo pauses in the middle of a run but doesn’t produce any error messages, just wait it out and it will resume the run after working out the problem.